DNA mismatch repair (MMR) plays a number of critical roles in eukaryotic cells including: 1) suppression of mutations that result from misincorporation errors during DNA replication as well as chemical damage to DNA and DNA precursors; 2) preventing genome rearrangements due to recombination between divergent DNA sequences; 3) repair of mispaired bases in recombination intermediates; and 4) DNA damage signaling linked to cellular responses such as cell cycle control and cell death. As a consequence, MMR defects cause increased rates of accumulating mutations and altered recombination events resulting in a characteristic genome instability signature as well as increased resistance to killing by some DNA damaging agents. Because MMR is defective in both inherited and sporadic cancers, understanding MMR will impact human health for a number of reasons: 1) a better understanding of the genetic consequences of MMR defects will impact the development of clinical tests for the MMR status of patients and tumors; and, 2) MMR defects result in resistance to many chemotherapeutic agents so understanding MMR and MMR defects could lead to improvements in cancer therapy. The goal of this proposal is to use Saccharomyces cerevisiae to study the biochemical and genetic mechanisms of the eukaryotic MutS and MutL homologue-dependent MMR pathways. The following lines of experimentation will be carried out: 1) genetic studies will identify MMR genes and proteins that function in redundant and overlapping MMR sub-pathways, which will guide biochemical studies of MMR; 2) cell biology and genomic approaches will be used to elucidate the mechanism of coupling of MMR to DNA replication; 3) biophysical and genetic approaches will define the protein-protein interactions and conformational changes that underlie the specificity of MMR; 4) partial and complete MMR reactions will be reconstituted in vitro using purified proteins to study the mechanisms of MMR; and 5) collaborative mouse model studies will be continued to extend insights from studies with S. cerevisiae to mammalian systems, with a particular focus on studying polygenic interactions and redundant MMR sub-pathways. The ultimate goal of these experiments is to understand the biochemical mechanisms of MMR and how cells utilize MMR to prevent mutations and genome rearrangements. A key feature of these studies is the use of S. cerevisiae to explore questions raised by the genetics of human cancer susceptibility, and collaborative mouse studies to explore the broader implications of results developed in S. cerevisiae. As a consequence, these studies will provide insights into the genetics of human cancer susceptibility and the biology of MMR defects in human cancers in addition to providing a basic understanding of MMR mechanisms.